Two-photon laser-scanning microscopy (TPLSM) is a critical tool for basic and biomedical neurobiology. It allows high-resolution, high-contrast three dimensional imaging within intact brain tissue. TPLSM allows detailed study of brain structure and function at the level of molecules, synapses, neurons, and circuits. Increasingly TPLSM is used to study brain disorders, such as stroke, epilepsy, Alzheimer's disease, Parkinson's disease, and spinal cord injury. This proposal aims to support and extend Scan-image - free, open-source software for controlling laser scanning microscopy instruments for neurobiology. Scan-image allows a rapidly growing number of scientists, technologists, and small manufacturers to develop home-built and/or specialized microscopes for brain research. Such standalone software for TPLSM control enables widespread and innovative uses of TPLSM for neurobiology. The flexibility to develop and deploy specialized microscope systems is increasingly needed to enable and propagate advanced approaches such as large-scale brain volume imaging, targeted manipulation of neurons via optogenetics, and imaging in awake, behaving animals. It is proposed to broaden the impact of TPLSM approaches and instruments used for neurobiology by: 1) integrating control and measurement of electrophysiology and behavioral data streams with Scan-image; 2) supporting a wider range of TPLSM microscopy hardware under Scan-image; 3) supporting additional existing and new modes of TPLSM with benefit to neurobiology; and 4) providing proactive and reactive support for Scan-image to the fast-growing user community. The resource will be comprised of software developer time to be allocated and prioritized among these goals towards neuroscientist user requirements, in consultation with an international Steering Committee of leading neuroscientists. The recent rapid growth of TPLSM adoption and publications in neurobiology reflects the great current and projected scientific value of high-resolution imaging in intact brain tissue. Maintaining Scan-image as a cutting-edge, dynamic, and flexible tool for TPLSM instrument and experiment control will surely lead to a new wave of important developments and discoveries in brain science.